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. 2022 Aug 12;12:944819. doi: 10.3389/fcimb.2022.944819

Figure 3.

Figure 3

LgyLRV1+ triggered type I IFNs are necessary but not sufficient in order to induce iNOS in an auto- and paracrine manner. (A) WT, Tlr3-/- or Ifnar-/- BMDMs were infected with LgyLRV1+ or LgyLRV1- (MOI 10), stimulated with poly (I:C) (2 μg/ml), FSL-1 (10 ng/ml), IFN-β (500 U/ml), alone or in combination, or left untreated for 12h. Total cell lysates were analyzed by western blot for iNOS and γ-tubulin expression. (B, C) WT, Tlr3-/- or Ifnar-/- BMDMs were treated as in (A) for 48h. SNs were collected and levels of nitrites were measured using Griess assay. (D) WT BMDMs were infected with LgyLRV1- (MOI 10), treated with FSL-1 (10 ng/ml) alone or in combination. Except one, all conditions were treated with poly (I:C), with one of eight two-fold serial dilution concentrations ranging from 2 to 0.015 μg/ml. Upon 48h SNs were collected and levels of nitrites were measured using Griess assay. (E) scheme depicting experiments performed in (F, G). (F, G) WT BMDMs were infected with LgyLRV1+ or LgyLRV1- (MOI 10), stimulated with poly (I:C) (2 μg/ml), or left untreated. Upon 6h, SNs were collected, filtered with 22 um filter, supplemented with FSL-1 (10 ng/ml) and added to the fresh set of untreated WT and Ifnar-/- BMDMs. Upon 24h, SNs from treated BMDMs were collected and levels of nitrites were measured using Griess assay (G). Total cell lysates were analysed by western blot for iNOS and γ-tubulin expression (F). Data show mean ± SD from a representative of three independent experiments (B, C, F). Representative blots are shown from at least three independent experiments in (A, F). the following: ****p < 0.0001, n.i. – non-infected. n.t. – non-treated. n.d. - not detected.